The present invention relates to transgenic and non-transgenic plants that produce oil having high levels of saturated fatty acids and medium to high levels of oleic acid. More specifically the invention relates to a sunflower plant, seed and the extractable oil having increased stearic acid content and increased oleic acid content relative to the normal sunflower oil level of stearic acid and oleic acid.
Sunflowers (Helianthus annuus) are cultivated for the seeds. Seeds have two principal uses. Seeds are used as confectionery seeds but much more importantly for extraction of oil formed within the sunflower seed. In the endogenous oil are a number of fatty acids. The total fatty acid content is given for 3 types of sunflowers: normal, high oleic and high palmitic. These contents are listed below. The saturated or unsaturated fatty acid is given as a percentage of weight relative to the total fatty acid content.
As table 1 indicates a number of different fatty acid distributions in sunflower oil are known. The table gives the wild type or normal sunflower oil as having a fatty acid distribution of the principle saturates of C16:0 levels of 5%-7%, C18:0 levels of  less than 10%, and of the principle unsaturates of C18:1 levels of 17%-20% and C18-2 levels of 50%-70%. Presently, high oleic and high palmitic are known sunflower oils with changed saturate, unsaturated fatty acid profiles.
The industry has been using both transgenic approaches and mutation approaches to alter the saturate, unsaturated fatty acid profiles in the fatty acid biosynthesis pathway. The percentage of saturated and unsaturated fatty acid present in an altered oil reflect the oil""s chemical and physical traits. The chemical and physical traits of the oil are altered to form either more useful oil for the industry and/or a healthier oil for the end user.
The last twenty years of sunflower research has produced a healthier oil in high oleic sunflower oil. This research has resulted in the commercial availability of sunflowers having high concentrations of oleic acids (C18:1) percentage by weight based in the total fatty acid content of the sunflower oil. Early Russian research into increasing oleic levels in sunflower seeds used mutagenesis to form Pervenets sunflowers having elevated oleic acid levels. Such oleic sunflower material is available to the public. Much, if not all, of the high oleic sunflower germplasm available today is the descendants of the early Russian lines resulting from that research. Commercially available high oleic sunflower seed includes for example, sunflower variety, Pioneer Hybrid 6661, which is marketed as producing a seed storage oil having a fatty acid composition that includes 85% oleate.
The availability of other high oleic sunflower seed is listed within a number of patent documents. For example, there is intellectual property describing high oleic lines which is believed to be based on the Russian Pervenets sunflowers in U.S. Pat. No. 4,627,192 and examination certificate B1 U.S. Pat. No. 4,627,192, issued Oct. 17, 1995, and U.S. Pat. No. 4,743,402 and Re-examination certificate B1 U.S. Pat. No. 4,743,402, issued Apr. 8, 1997 to Flick. These patents list a number of sunflower varieties that are commercially available for breeding purposes that can be licensed under the Fick patents through a company called A. C. Humko. Additionally, the Fick U.S. Pat. No. 4,627,192 indicates that oleic seeds of Sigco 41A, 41b, 853R, 4117b, 273W, 416R are available from the Lubrizol Corporation, 29400 Lakewood Blvd., Wickliffe, Ohio (USA) 44092.
In addition to the research, which resulted in increased oleic acid in sunflower oil, there has also been research on increasing the overall industrial usefulness of sunflower oil usually addressed by increasing the level of the saturated fatty acids in oil. Increasing the saturated fatty acids produces an oil that is more suitable for use in the production of margarine, shortening, other food products, and cosmetics by decreasing the need for substantial amounts of hydrogenation.
Some of this type of research is outlined in Osorio et al., in Crop Sci. 35:739-42 (1995). This article describes sunflower seeds developed by traditional breeding and mutagenesis to produce seeds with a high stearate content. This type of research is also outlined in PCT application number EP95/00369 which is entitled xe2x80x9cSunflower seeds and oil having a high stearic acid contentxe2x80x9d. This application teaches, as its name implies, a sunflower oil with increased stearic acid content. One way to obtain this oil is by treating parent seed with a mutagenic agent to induce one or more mutations in the stearic acid biosynthesis pathway. This process resulted in an increased production of stearic acid in the sunflower oil in a range between 12% and up to 35% by weight of stearic acid related to the total amount of fatty acid in the oil. High stearic acid producing seeds discussed in this patent are under deposit in the American Type Culture Collection (ATCC). Sunflower seeds identified as xe2x80x9cCAS-3xe2x80x9d have an average stearic acid content of 25% by weight, related to the total amount of fatty acids in the oil. These seeds were deposited on Dec. 14, 1994 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A., under deposit accession number ATCC 75968. Sunflower seeds identified as xe2x80x9cCAS-4xe2x80x9d having an average stearic acid content of 15% by weight, related to the total amount of fatty acids in the oil, were deposited on Dec. 14, 1994 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A., under deposit accession number ATCC 75969.
This application PCT/EP95/00369 suggests that oil from high stearic lines could be combined with oil from high oleic lines for certain industrial uses. Unfortunately, although this combination of two oils is useful in many instances, there remains a need for a seed that produces high levels of both stearic and oleic fatty acids in the same oil, particularly since the levels of linoleic acid produced by these stearic acid lines may tend to produce a less desirable profile of fatty acids than would be produced by a hybrid producing high stearic, high oleic acid.
Traditional breeding and mutagenesis has not been the only tool used to form seeds producing oil with different fatty acid profiles. Increases in stearic acid in oil bearing plants have also been addressed by the introduction of transgenes in to the germplasm, to alter the fatty acid biosynthesis pathway of the vegetable oil. The fatty acid biosynthesis in vegetable oil, but more particularly sunflower oil, includes the biosynthesis of basically two saturates palmitate, stearate and two unsaturates oleate and linoleate. To give a simplified description of the biosynthesis pathway, it is sufficient to say, that palmitate (C16:0) is by enzymatic action chemically modified to form stearate (C18:0), which by enzymatic action is modified to produce oleate (C18:1), that is further modified to form linoleate (C18:2), some minor amounts of araquic (C20.0) and behenic (C22:0) acids are also formed from stearate. In oilseeds the stearoyl-ACP desaturase is the enzymatic action which introduces the first double bond on stearoyl-ACP to form oleoyl-ACP. Thus, this is an enzyme that assists in the determination of the unsaturation in the C18 length fatty acids.
In U.S. Pat. No. 5,443,974 the inhibition of canola enzyme stearoyl-ACP desaturase was described. The stearate levels were increased but the levels of palmitate were basically unaffected Inhibition of the plant enzyme stearoyl-ACP desaturase in canola was also reported by Knutzon et al., Proc. Nat""l Aca., Sci. USA 89:2624-28 (1992). These results showed an increase in the level of stearate produced in the canola seed. The research also showed that inhibition by antisense in seeds of canola and soybean, respectively showed increased stearate. When a plasmid containing a gene encoding for stearoyl-ACP desaturase was placed in canola, this inhibition resulted in both an increase in stearic acid and unfortunately a reduction in the oleate. However, in the soybean this inhibition of stearate resulted in a less dramatic reduction of the oleate. This slower decrease in oleate however may have been a function of the small initial levels of oleate in the soybean. The fatty acid pathway in most oilseed plants appears to be resistant to maintaining both oleic and stearic at elevated levels.
Additionally, Pioneer Hi-Bred International, Inc. has increased the levels of both stearic acid and palmitic acid in sunflowers through the inhibition of the plant enzyme stearoyl-ACP desaturase. This research was surprising in light of other transformations in other plants that indicated that this enzyme would not affect palmitic acid levels only stearic acid levels. Unfortunately, palmitic oil is not viewed as being a healthy oil. This research is indicated in PCT/US97/01419.
Pioneer Hi-Bred International, Inc. taught in PCT/US96/09486 that sunflower oil levels of both palmitic and oleic acids could be increased. Sunflower seeds having increased levels of palmitic 21-23% and oleic 61% were deposited in the ATCC as 93PMOL040G and 93PMOL040F in the American Type Culture Collection 12301 Parklawn Drive, Rockville Md. 20852, U.S.A. having Accession No. 97159 and 97158 respectively. The patent application teaches a sunflower oil that is liquid at room temperature. But the increased palmitic fatty acid level in the Pioneer invention, is alleged to allow the oil to be used in shortening and in margarine with relatively low level of hydrogenation, which leads to a relatively low level of trans-fatty acids in the resulting product. However the commercial value may be questioned as palmitic oil is not viewed as being a very healthy oil. Thus margarine made with this oil may be substantially less desirable than margarine made with a healthier combination of fatty acids (i.e. stearic acid).
There remains a need for a sunflower oil which is healthy and useful for industrial purposes. There is a need for a sunflower oil that has a balance of good saturates and good unsaturates. There also remains a need for a sunflower oil that is high in unsaturates but has sufficient saturates to be used for margarines or hardstock without high levels of hydrogenation which lead to no trans-fatty acids in the resulting product. There remains a need for a sunflower plant that can produce seed containing oil which is high in oleic acid and in stearic acid with reduced linoleic levels.
It is an object of the present invention to provide novel sunflower plants which form seeds which contain an endogenous oil wherein the levels of stearic acid (C:18) and oleic acid (C:18:1) are provided in an atypical combination.
It is further an object of the present invention to provide sunflower plants seeds and oil obtainable from the seeds that has an oleic content of more than 40% by weight of oleic acid and a stearic acid content of more than 18% by weight.
It is further an object of the present invention to provide sunflower seed and lines which when crossed form hybrids having the genetic characteristics to yield high levels of oleic acid and high levels of stearic acid content in the oil as compared to oil obtained from wild type seeds.
It is an object of the present invention to provide F1 hybrid seeds having the genetic characters to yield plants which form seeds characterised by high levels of oleic acid and high levels of stearic acid content as compared to oil obtained from wild type seeds.
The invention further relates to sunflower plants produced from seeds according to the invention that can produce parent lines with high stearic acid and high oleic acid levels in their oil. This applies to the seeds and the progeny therefrom obtained after crossing the mutants of the invention with each other or with other germplasm having a desirable characteristic.
It is another object of the present invention to produce sunflower plants that may be used in breeding programs aimed at the production of open pollinated or inbred or hybrids having acceptable agronomic traits that are useful in the field environment.